Structural Biochemistry/Proteins/Preparation of Monoclonal Antibody

Monoclonal antibodies are a homogeneous collection of antibodies that all bind to the same antigenic determinant. They were first prepared by Cesar Milstein and Georges Kohler.

Similar to working with impure proteins, working with impure antibodies(polyclonal antibodies) made it difficult to interpret data and to understand function. Because their specificity was not known, immunological methods (e.g. ELISA, Western Blotting) were not able to be applied.

The ideal method of obtaining monoclonal antibodies would be to simply isolate the desired antibody producing cell. However, the problem with that is that antibody producing cells isolated from the organism don't live very long. The solution to this problem would be to use a cancer cell (myeloma cell).

Multiple myeloma is a disorder of antibody producing cells characterized by the uncontrollable division of a mutated cell. Each time this cell divides, it produces an exact copy of itself. Because cell division is not being regulated, a large aggregation of cells are quickly formed. All of these cells are identical to one another, and are thus referred to as clones. Milstein and Kohler hypothesized that if a cancer cell of this type could be fused with an antibody producing cell, they could have a potentially infinite source of monoclonal antibodies.

Monoclonal antibodies are produced by the process outlined in the flowchart above. A mouse is first injected with an antigen that is known to stimulate the production of the desired antibody. After approximately three weeks, the spleen of the mouse is removed. Plasma cells from the spleen are fused in vitro with myeloma cells, giving rise to a colony of hybrid cells (hybridoma cells). These hybridoma cells are capable of producing an indefinite amount of the antibody obtained from the spleen.

How to obtain monoclonal antibodies

A mixture of hybridoma cells is often formed. This mixture must be separated in order to obtain a homogeneous collection of antibody producing cells. An assay that tests for specific antibody-antigen interactions can be used to achieve separation. The assays are repeated and the collection of cells is continually subdivided until a pure cell line is obtained. It is good to note that the spleen also contains numerous other cells that produce antibodies for different antigens. Purification is necessary is order to find the one type of hybridoma cell that produces the desired antibody. The isolated cells can either be grown in a culture or injected into a mouse in order to procure the desired antibody. Another option is to freeze the cells for later use.

The advantage of monoclonal antibodies is that they are identical antibodies with the same affinity for the same area of the same antigen, thus allowing better purification results. Polyclonal antibodies, on the other hand, are a mixture of different antibodies each recognizing a different site (epitope) of the same antigen.

Antibodies have the ability to bind to two antigens. This is possible since any antibody has the ability to bind to two antigens and can be recognized by many antibodies (monoclonal and polyclonal) as far as there is still exposed surface where the antibodies could bind.